Developing B and T lymphocytes assemble their antigen receptor genes through a process of somatic gene rearrangement known as V(D)J recombination, in which a complex of Rag1 and Rag2 proteins (RAG) induces DNA double strand breaks (DSBs) that are repaired by Non-Homologous End Joining. Errors during V(D)J recombination can cause translocations involving antigen receptor loci, in some cases leading to cancer. It has recently been determined that DSBs induced by RAG or by ionizing radiation (IR) in pre-B cells suppress expression of Rag1 and Rag2 mRNA and protein. Furthermore, RAG DSBs have been demonstrated to inhibit V(D)J recombination in pre-B cells. Because the existence of multiple DSBs in a cell greatly increases the risk of translocation, suppression of RAG DSBs in the presence of other DSBs may help preserve genomic stability in developing lymphocytes. Thus, the central hypothesis of this proposal is that DSBs induced in developing lymphocytes actively inhibit V(D)J recombination by suppressing RAG expression, thereby mitigating risk of these DSBs forming oncogenic translocations involving antigen receptor loci. Preliminary data show that DSBs induced in pre-B cells by IR activate the DNA-damage response protein Ataxia Telangiectasia Mutated (ATM), leading to decreased mRNA and protein levels of Rag1 and Rag2. Aim 1 of this proposal will test the hypothesis that DSBs act through ATM to inhibit Rag1 and Rag2 transcription, while ATM-independent signals promote degradation of RAG proteins. Transcription and degradation rates of Rag1 and Rag2 mRNAs in cells with or without DSBs induced will be compared, as will Rag1 and Rag2 protein phosphorylation and degradation. Aim 2 will test the hypothesis that inhibition of Rag1/Rag2 transcription is the primary mechanism by which DSBs inhibit V(D)J recombination. To determine whether sustained RAG expression is sufficient to cause excessive RAG-mediated DNA cleavage, ex vivo pre-B cells that are unable to repair RAG breaks will be infected with Rag1/Rag2-encoding lentiviruses and frequencies of RAG-mediated DSBs will be assessed. To determine whether the uninhibited V(D)J recombination in the presence of other DSBs promotes the formation of oncogenic antigen receptor translocations, bone marrow chimaeras will be made in which either all hematopoietic cells, or specifically B or T cells, ectopically express Rag1 and Rag2. These mice will be monitored for defects in lymphocyte development, the presence of lymphocytes with translocations at antigen receptor loci, and the development of lymphoma or leukemia. The successful completion of these studies will provide a deeper understanding of how developing lymphocytes regulate V(D)J recombination, a process with potential to cause genomic instability and cancer. The results should provide insight into the initiating events of lymphoid malignancies involving antigen receptor translocations and suggest new factors that predispose to such cancers.